KINETIC-ANALYSIS OF ESCHERICHIA-COLI RIBONUCLEASE HI USING OLIGOMERICDNA RNA SUBSTRATES SUGGESTS AN ALTERNATIVE MECHANISM FOR THE INTERACTION BETWEEN THE ENZYME AND THE SUBSTRATE/

Escherichia coli ribonuclease HI mainly recognizes the DNA/RNA hybrid regions preceding the cleavage site. To understand the interaction bet ween the enzyme and the substrate in more detail, the kinetic properti es of the enzyme, as well as its variant with mutations in the basic p rotrusion, were studied using a series of oligomeric DNA/RNA hybrids a s substrates. These substrates were prepared by hybridizing a 12-b RNA (5'-CGGAGAUGACGG-3') with DNA oligomers varying in size and sequence. The 12-b RNA hybridized to the complementary 12-b DNA was primarily c leaved at A9-C10. Since an increase in the length of the RNA between t he cleavage site and the 5' end of the DNA/RNA hybrid, achieved using a longer DNA/RNA substrate, did not seriously affect the kinetic param eters of the enzyme, the 12-bp DNA/RNA hybrid seems to be large enough to contact the entire substrate-binding site of the enzyme. The kinet ic data presented here suggest that the DNA residues complementary to the RNA residues located six or seven residues upstream from the cleav age site interact with the basic protrusion of the enzyme, regardless of whether or not it is hybridized to the RNA. strand. Such an interac tion is permitted only when the conformation of either the enzyme or t he substrate, or both, is changed upon binding.